The goal of this proposal is to exploit oral vaccination with a novel HIV envelope (HIVenv)-loaded pollen grain (PG) formulation to elicit a rapid immune response comprising of HIVenv-specific immunological memory in Natural Killer (NK) cells that directly kill HIV infected cells in the gastrointestinal (GI) mucosa, to prevent HIV infection. NK cells are an essential anti-viral weapon of the immune system; they respond quickly to infection, kill infected cells without prior activation, engage multiple targets simultaneously, and secrete large amounts of anti-viral cytokines, such as interferon gamma. Further, NK cells also crosstalk with B cell-mediated Ab- responses through antibody-dependent cell-mediated cytotoxicity (ADCC), a rapid NK cell-mediated lytic function that only requires tagging of target cells by binding Abs that may not even be neutralizing. If these unique NK cell-mediated effector functions could be enhanced through vaccination, and induced rapidly at the site of HIV entry, it may be possible to prevent the establishment of a systemic HIV infection. In support of this postulate, Dr. Paust (PI) and colleagues have recently demonstrated that contrary to conventional paradigm, subsets of NK cells mediate Ag-specific immunological memory to HIV-envelope. However, reliable elicitation of strong mucosal immunity through vaccination remains a challenge. Recently Dr. Gill (Co-I) has pioneered a unique approach that uses pollen grains (PGs) for oral vaccination. PGs, due to their natural toughness, survive the harsh environment of the stomach, and safely ferry antigens (Ags) to the intestine, where they are persorbed cross the intestinal epithelium. Using these methods, we generated data, which show that oral vaccination with ovalbumin (OVA)-loaded lycopodium spores (LS-OVA) results in (i) OVA-specific Abs in serum and fecal matter, (ii) OVA-specific memory NK cells, which degranulate, kill, and mediate ADCC upon in vitro restimulation, and (iii) memory NK cell recruitment to the mucosa upon oral challenge. We further found that HIVenv elicits recall responses in human NK cells in vitro, and in vivo. Based on these considerations, we hypothesize that human NK cells with antiviral activity against HIV represent a potential effector cell population able to prevent systemic HIV infection, and that oral LS-env vaccination elicits HIV-specific memory NK cell responses. We propose to determine the efficacy of a novel oral LS-env vaccine for the induction of long-lived HIV-specific mucosal NK cell-memory responses in humanized mice, and to assess the efficacy of NK cell memory for the prevention of HIV infection. The demonstration of memory NK cells with activity against HIV in human NK cells, and the ability to induce these responses by vaccination would be highly significant, as it would identify a novel adaptive lymphocyte subset that can be targeted by vaccines and directed against HIV.